A vast array of novel strategies has been introduced over the past 50 years for the treatment or early and advanced stage cancer. Early attempts at cancer immunotherapy focused on IL-2 or other T cell?activating cytokines that were intended to expand the frequency or activation state of T cells specific for tumor-associated antigens (TAAs). These activated T cells were expected to infiltrate the tumor and lead to the specific destruction of tumor cells. Typically, an effective T cell activation depends on the presentation of TAAs by antigen-presenting cells (APCs). While a number of potential TAAs have been reported in literature, the clinical applicability of many of these antigens are often not properly followed with specific immunological studies. Advances in TAA-based immunotherapy were hampered by absence of reliable animal models and inefficient vaccine approaches, in particular, poor adjuvants and delivery systems. Furthermore, tumor cells have been recently reported to employ CD4+CD25+ T regulatory cells (Treg) to evade specific host-mediated anti-tumor responses. The specific aims of our current research included the (1) development of cancer vaccines which target/deliver antigens to various subsets of APCs, specifically immature DCs, (2) development of chemotoxins (chemokines fused with toxins) for the preferential depletion or modulation of ?bad? cells resulting in the enhancement of anti-cancer responses and (3) the development of novel chemoattractant peptide-based vaccine carriers and their immunomodulatory functions. 1). The ideal vaccine carrier should be capable of delivering the target antigen to antigen-presenting cells (APC), while, at the same time, inducing APC and lymphocyte recruitment and activation. In this respect, the chemoattractant peptides may fulfill these requirements, in that they can both target and attract APC, and in some cases (e.g., murine beta-defensin 2) can activate cells via TLR-4. We have reported that non-immunogenic antigens were rendered immunogenic by targeting them to APCs via chemokine receptors. In vivo studies have suggested that, in order to be effective, vaccines require the targeting of immature, but not mature, DCs. Recently, we have demonstrated that antigens delivered to chemokine receptors are efficiently internalized, processed and presented utilizing both MHC class I and II processing and presentation pathways. The chemokine-fused antigens were carried to early/late endosomal and lysosomal compartments through a clathrin-dependent process and subsequently delivered them to the cytosol for proteasomal degradation, thus facilitating efficient cross-presentation thorough the TAP-1-dependent process. Typically, approaches that target various endocytic cell surface receptors are known to increase the efficiency of antigen presentation by 100- to 10,000-fold. In this respect, chemokine receptor targeting is also quite efficient in that only nmol (ng/ml) quantities of tumor antigens fused with chemokine are required to mount an effective response. Therefore, this is a very simple and potent approach, which has been reproducibly shown to elicit protective and therapeutic antitumor immunity in several syngeneic murine tumor models. In addition, we have initiated testing various strategies for the development of universal formulations for treatment of several malignant diseases. The formulation is based on use of the embryonic antigen, OFA-iLRP, a highly conserved the 37-kDa oncofetal antigen immature laminin receptor protein, which was reported to be expressed in number of human malignancies. Since OFA-iLRP is expressed in renal carcinoma cells, we also hoped that this formulation might enable us to treat renal carcinomas, a deadly disease with no established tumor antigen markers to date. Preliminary data has suggested that chemoattractant-based DNA vaccines encoding OFA-iLRP can elicit therapeutic antitumor immunity against syngeneic mouse tumors that express OFA-iLRP. 2). Chemokine-based vaccines typically result in up to 50% tumor free mice when utilized for treatment of established syngeneic tumors. The inability of these vaccines to elicit 100% tumor eradication might be due to several factors, including antigen escape and involvement of Treg cells. Treg cells migration, particularly skin-homing, is controlled by chemokines, which signal via binding to differentially expressed G-protein coupled chemokine receptors, such as CCR4 and CCR8. Therefore, we have proposed a novel strategy for transient depletion of Treg cells may promote preferential induction of long lasting protective cellular responses. To achieve this, we produced chimeric proteins, designated as ?chemotoxins? (chemokine + toxin), which exist as inert moieties unless delivered to the cytosol of cells. We have shown that CCR4-targeting chemotoxins efficiently killed Treg cells and augmented tumor antigen specific T cell responses in vitro. Currently, we have initiated testing the in vivo relevance of this strategy. On the other hand, we have also shown that chemotoxins can be also used for eradication of tumors that preferentially express chemokine receptors, such as T cell lymphoma and adult T-cell leukemia/lymphoma (ATLL) that abundantly expressed CCR4. For example, CCR4-targeting chemotoxins were able to efficiently kill human T lymphoblastoid CCRF-CEM cells (CEM) in vitro within 1-2 days of treatment (IC50 of 3-8 nM), and eradicate s.c. established CEM tumors in NOD-SCID mice leaving only scared tissues at the site of injections. While mock treatment or TARC-OFA injections failed to protect mice, 100% of mice treated with chemotoxin survived and did not demonstrate palpable tumor during the observed period. Together, these results suggest that the use of chemotoxins may be valuable for the treatment of human T cell malignant diseases when a patient?s immune system is severely immunocompromised by disease or age. 3). An important part of our research is dedicated to the development of ?more efficient, potent and safer? carriers that would deliver weakly-immonogenic antigens to APCs to render them immunogenic. We have explored the use of viral-derived chemokines as carriers to circumvent vaccine-induced anti-host chemokine immune responses. Our studies have revealed that viral chemokines can also be efficiently utilized for vaccine delivery. Moreover, we have examined a novel and as-of-yet uncharacterized function of the antimicrobial peptides. This work was initiated by our recent report demonstrating that murine beta-defensin 2 (mDF2b) not only recruited immature DCs through the use of the chemokine receptor CCR6, but also activated their maturation through the TLR-4. To date, none of characterized human beta-defensins were capable of activating DCs, and it is unclear whether a functional homolog of mDF2b is present among more than 30 hypothetical human beta-defensin genes reported. During our studies, we have serendipitously found that mDF2b may also play a role in the elimination of activated APCs. Moreover, we demonstrate that mDF2b promoted a caspase- independent, TLR4/MyD88-dependent signaling cascade resulting in TNFa/TNFR2- mediated apoptosis and necrosis of APCs. Overall, the use of chemokines as vaccine carriers is an efficient and simple strategy to elicit both humoral and cellular responses. Taking advantage of chemokine redundancy, we have also demonstrated that viral chemokine fusions were equally potent in inducing protective immunity in vivo, providing a possible strategy to circumvent hypothetical, vaccine-induced anti-host chemokine autoimmunity. Moreover, we have developed a novel strategy for depletion of unwanted subsets of cells, at will, using chemotoxins.